There is a need to develop new agents and technologies to image and quantitate molecular signatures in vivo. The main rationale for this is two-fold: a) to allow the earlier detection of disease based on molecular abnormalities and b) to image specific biomarkers during treatment (e.g. the imaging of a protease duringprotease inhibitor treatment). In prior research we have a developed a number of"smart" sensing optical molecular probes that change their fluorescent properties after target interaction. One of the main challenges so far has been to detect and quantitate these activatable molecular beacons in vivo. Based on an optical tomographic method we have now built the ftrst prototype small animal imaging system that is capable of detecting fluorescence activation in deep tissues (fluorescence mediated tomography, FMT). The overall goaJ of this application is to improve on the performance of this system, quantitate, validate and correlate the data and then expand the technology to more sophisticated and more sensitive measurements. During the R21 phase of the grant application (year 01), we will further optimize the system components of the prototype imaging system. During the subsequent R33 phase of the application, we will pursue the following aims: 1) design and build a larger source-detector array, higher sensitivity and lower noise FMT system, suited for whole-body and higher resolution imaging, and test it with appropriate mouse/molecular probes models; 2) integrate time-resolved technology into the FMT system to improve reconstructions and quantitation of fluorescence; 3) design an appropriate MR-compatible optical tomographic system. We will address the following clinically relevant questions: 1) can the technology be applied to detect the activation of molecular beacons in early/small tumors, 2).can the systems be used for quantitative measurements of specific enzymes in vitro and in vivo and 3) can the efficacy of protease inhibitors be imaged at the molecular level? The approach, if successful, is expected to have broad applications to a variety of novel biologic, immunologic, and molecular therapies designed to control and eradicate cancer.